1. Field of the Invention
The present invention relates to a method for synthesis of nucleic acids, especially to a method for synthesis of nucleic acids by means of a polymerase chain reaction (hereinafter abbreviated as a PCR).
2. Description of the Related Art
A PCR method is a procedure capable of amplifying an intended DNA fragment as much as several hundred thousand-fold by repeating a process comprised of dissociation of a DNA strand into single strands, binding of primers with sandwiching a particular region of the DNA strand, and a DNA synthesis reaction by the action of a DNA polymerase. The PCR method is described in Japanese Laid-open Patent Publication No.S61-274697 which is an invention by Mullis et al.
A PCR procedure can be used as a highly sensitive method for analyzing nucleic acids in various samples, and particularly it can be used in analysis of nucleic acids in a sample derived from an animal body fluid. The PCR procedure is therefore used for such a purpose of diagnosis or monitoring of an infection, a hereditary disease, and a cancer. The PCR procedure is also suited to DNA typing tests for a transplantation, a paternity test, medical treatments based on an individual genetic information, and the like. For these purposes, a peripheral blood is often selected as a test object.
One drawback of the PCR procedure is that the reaction is inhibited by pigments, proteins, saccharides, or unknown contaminants. Namely, many DNA polymerases including TaqDNA polymerase derived from Thermus aquaticus, a typical thermostable DNA polymerase, are widely known to allow the PCR to be inhibited potently by even a trace amount of living body-derived contaminants existing in the PCR reaction solution. Therefore, the PCR procedure requires a process in which a cell(s), a protozoan (protozoa), a fungus (fungi), a bacterium (bacteria), a virus(es) and the like (hereinafter referred to as a gene inclusion body) are isolated from a subject and then nucleic acids are extracted from the gene inclusion body prior to a DNA amplification. Such process has conventionally been a procedure in which the gene inclusion body is decomposed using an enzyme, a surfactant, a chaotropic agent, or the like, and then nucleic acids are extracted from the decomposed product of the gene inclusion body using, for example, phenol or phenol/chloroform. Recently, an ion-exchange resin, a glass filter, or a reagent having an effect of agglutinating proteins is used in the step of the nucleic acid extraction.
It is difficult, however, to completely remove impurities by purifying nucleic acids in a sample using these procedures, and furthermore, an amount of nucleic acids in a sample recovered by these purification procedures often varies among experiments. For these reasons, a subsequent nucleic acid synthesis may sometimes be unsuccessful, especially when a content of the intended nucleic acid in the sample is low. In addition, these purification procedures involve complicated manipulations and are time-consuming, and there is a high opportunity for contamination during the procedures. Therefore, a simpler, more convenient and effective method of a sample pretreatment is desired in order to solve these problems.
Thus, an object of the present invention is to provide a novel method for removing nucleic acid synthesis inhibitory substances and thereby amplifying a nucleic acid in a sample efficiently.
The present inventor found that nucleic acid synthesis inhibitory substances in a biological sample can be removed by bringing the substances into contact with an insoluble polymer of a polyanion, and thus arrived at the present invention.
The present invention is a method for synthesis of nucleic acids to amplify an intended nucleic acid in a sample which comprises bringing the sample in advance into contact with an insoluble polymer of a polymer compound having a repeating structure containing at least one anion (polyanion) and/or a salt thereof (hereinafter collectively referred to as a polyanion).
The present invention is the method for synthesis of nucleic acids wherein the polyanion is a polymer compound having a repeating structure containing at least one sulfate group (polysulfate) and/or a salt thereof (hereinafter collectively referred to as a sulfated polymer).
The present invention is the method for synthesis of nucleic acids wherein the sulfated polymer is selected from the group consisting of sulfated polysaccharides and salts thereof (hereinafter collectively referred to as a sulfated polysaccharide).
The present invention is the method for synthesis of nucleic acids wherein the sulfated polysaccharide is selected from the group consisting of heparin and a salt thereof, and dextran sulfate and a salt thereof.
The present invention is the method for synthesis of nucleic acids wherein the sample is a living body-derived sample itself.
According to the present invention, by conducting a simple and convenient treatment in which a sample, such as serum or plasma, containing a lot of PCR inhibitory substances is brought in advance into contact with an insoluble polymer of a polyanion, it becomes possible to directly amplify an intended nucleic acid efficiently without undergoing a process of isolating and purifying nucleic acids from the sample. It becomes also possible by the present invention to perform procedures for nucleic acid synthesis more simply, conveniently and rapidly, and thereby reduce the opportunity for contamination.